Nucleic acids encoding interleukin-12P40 variants with improved stability

ABSTRACT

Modified interleukin-12 (IL-12) p40 polypeptides are disclosed. The modified polypeptides have alterations in the IL-12p40 subunit to eliminate the protease site between positions Lys260 and Arg261. The modified IL-12p40 polypeptides according to the invention have improved stability compared to wild-type mature human IL-12p40 polypeptides.

CROSS-REFERENCE TO RELATED APPLICATION

This application is a divisional application of U.S. application Ser.No. 11/647,661, filed Dec. 29, 2006, issued as U.S. Pat. No. 7,872,107on Jan. 18, 2011, which claims priority to and the benefit of U.S.Provisional Patent Application No. 60/755,382, filed Dec. 30, 2005, theentire disclosures of each of which are incorporated by referenceherein.

FIELD OF THE INVENTION

This invention relates generally to IL-12p40 proteins, including fusionproteins containing IL-12p40, modified to improve their stability. Inparticular, the IL-12p40 proteins of the invention remove a proteolyticsite in the region of Lys260 and Arg261 in the p40 subunit.

BACKGROUND OF THE INVENTION

Interleukin-12 (IL-12) is an inflammatory cytokine that is produced inresponse to infection by a variety of cells of the immune system,including phagocytic cells, B cells and activated dendritic cells(Colombo and Trinchieri (2002), Cytokine & Growth Factor Reviews, 13:155-168). IL-12 plays an essential role in mediating the interaction ofthe innate and adaptive arms of the immune system, acting on T-cells andnatural killer (NK) cells, enhancing the proliferation and activity ofcytotoxic lymphocytes and the production of other inflammatorycytokines, especially interferon-γ (IFN-γ).

IL-12 is a heterodimeric molecule composed of an α-chain (the p35subunit, IL-12p35) and a β-chain (the p40 subunit, IL-12p40) covalentlylinked by a disulfide bridge to form the biologically active 74 kDaheterodimer. Amino acid sequences of IL-12p35 and IL-12p40 of a mature(wild-type) human IL-12 are depicted in FIGS. 1 (SEQ ID NO:1) and 2 (SEQID NO:2), respectively.

Interleukin-23 (IL-23) is a disulfide-bridged heterodimeric moleculeclosely related to IL-12, in that it has the same chain IL-12p40 asIL-12, but a unique a chain (the p19 subunit, IL-23p19) (Oppmann et al.,(2000), Immunity, 13: 715-725). Like IL-12, IL-23 is produced byphagocytic cells and activated dendritic cells, and is believed to beinvolved in the recruitment and activation of a range of inflammatorycells (Langrish et al., (2004) Immunol. Rev., 202: 96-105). The aminoacid sequence of IL-23p19 of a mature human IL-23 is depicted in FIG. 3(SEQ ID NO:3).

For immune cells to secrete biologically active IL-12 or IL-23heterodimers, concomitant expression of the α and β subunits in the samecell is required. Secretion by immune cells of the IL-12p35 or IL-23p19alone has not been observed, whereas cells that produce the biologicallyactive IL-12 or IL-23 heterodimer secrete the p40 subunit in free formin 10 to 100-fold excess over the heterodimer (D'Andrea et al. (1992),J. Exp. Med., 176: 1387-98, Oppmann et al. (2000), Immunity, 13:715-725). In addition, it has been observed in the mouse that, even inthe absence of an α subunit, cells may produce a biologically activeIL-12p40 homodimer (Hikawa et al. (2004), Neuroscience, 129: 75-83).

The presence of endogenous IL-12 has been shown to be necessary forimmunological resistance to a broad array of pathogens, as well as totransplanted and chemically induced tumors (Gateley et al. (1998), Annu.Rev. Immunol., 16: 495-521). IL-12 has been demonstrated to have apotent anti-tumor activity based upon the induction of IFN-γ and theactivation of effector cells such as CD8+ T-cells and NK cells (Brundaet al. (1993), J. Exp. Med., 178: 1223-30). As a result of itsdemonstrated anti-tumor activity, IL-12 has been tested in humanclinical trials as an immunotherapeutic agent for the treatment of awide variety of cancers (Atkins et al. (1997), Clin. Cancer Res., 3:409-17; Gollob et al. (2000), Clin. Cancer Res., 6: 1678-92; and Hurteauet al. (2001), Gynecol. Oncol., 82: 7-10), including renal cancer, coloncancer, ovarian cancer, melanoma and T-cell lymphoma, and as an adjuvantfor cancer vaccines (Lee et al. (2001), J. Clin. Oncol. 19: 3836-47).

For IL-12 or IL-23, production of the recombinant protein in itscorrectly folded and biologically active, heterodimeric form, requiresthe concurrent expression of both the α subunit and IL-12p40 in theproducing cell line. The purified recombinant protein, however, exhibitsa degree of heterogeneity resulting from proteolytic cleavage in theC-terminal region of the IL-12p40. The instability of the IL-12 or IL-23protein can give rise to problems in its production and clinical use asa therapeutic agent. Therefore, there is a need in the art for improvedrecombinant IL-12 or IL-23 variants that yield a homogeneous proteinmore resistant to proteolytic cleavage.

SUMMARY OF THE INVENTION

The invention provides variants of human IL-12 p40 subunits (p40variants) which have improved stability compared to wild-type IL-12 p40proteins. In these p40 variants, the C-terminal region, which isnormally sensitive to proteolytic cleavage, has been engineered to bemore resistant to digestion by proteases. Specifically, p40 variants ofthe invention include engineered amino acid alterations in the D3 domainaimed to avoid the creation of potential T-cell epitopes that could makethe variant proteins immunogenic and trigger antibody responses inhumans. As a result, p40 variants of the invention have improvedproperties as therapeutic agents over wild-type IL-12p40 proteins withregard to their production, formulation, and pharmacokinetics.

Accordingly, in one aspect, the invention provides a variant of a humanIL-12 p40 D3 domain (D3 variant), wherein the D3 variant has at least85% identity with a wild-type human IL-12p40 D3 domain and includes anamino acid alteration at one or more positions corresponding to residues258-266 of mature human IL-12 p40. Certain embodiments of the inventionare based, in part, on an appreciation that an amino acid alteration oralterations according to the invention have the particular benefit ofremoving the proteolytic site between Lys260 and Arg261.

According to the invention, the amino acid alterations to one or morepositions corresponding to residues 258-266 may be deletions,substitutions, or insertions. Moreover, amino acid substitutions thatreplace basic amino acids with non-basic amino acids can be used tocreate variants according to the invention.

In particular, D3 variants of the invention may include one or moreamino acid substitutions at positions selected from the group consistingof Lys258, Ser259, Lys260, Arg261, Lys263, Lys 264, Asp265, and Arg266.Such amino acid alterations can be used singly or in combination toinduce the structural and/or functional changes described above. Forexample, the D3 variant can incorporate one, two, three, four or more ofthe following substitutions: Lys258Gln, Ser259Asp, Lys260Ala, Lys260Asn,Lys260Gln, Lys260Gly, Arg261Ala, Arg261Asp, Arg261Thr, Lys263Gly,Lys263Ser, and/or Lys264Gly.

In some embodiments, the substitution is a position Lys260. Thesubstitution may replace Lys260 with a non-basic amino acid, forexample, Ala, Asn, Gln, or Gly. Further substitutions in addition toLys260 may occur at Ser259 and Arg261. Particularly, some D3 variants ofthe invention incorporate substitutions Ser259Asp, Lys260Asn, andArg261Thr. In a further embodiment, D3 variants of the inventionincorporate substitutions Ser259Asp, Lys260Asn, Arg261Thr and Lys264Gly,while optionally deleting Lys263 and Asp265. Alternately, a D3 variantof the invention incorporates substitutions Ser259Asp, Lys260Asn,Arg261Thr, and Lys264Gly while deleting Lys263, Lys264 and Asp265.

In other embodiments according to the invention, a D3 variant includinga substitution replacing Lys260 alternatively includes furthersubstitutions at one or more of Lys258, Ser259, Arg261, Lys263, andLys264. For example, in one embodiment, a D3 variant includes thesubstitutions Lys258Gln, Ser259Asp, Lys260Gln, Arg261Asp, and optionallyLys263Ser and Lys264Gly.

In further embodiments, in addition to substitutions at Ser259, Lys260,and Arg261, one or more of residues corresponding to Lys263, Lys264,Asp265, and Arg266 are deleted, while in another embodiment, one or moreof Lys263, Lys264, Asp265, and Arg266 are substituted with a non-basicamino acid. In a further embodiment, the substitution at Lys264 isLys264Gly and, optionally, Lys263 and Asp265 are deleted. Other D3variants of the invention incorporate substitutions Ser259Asp,Lys260Asn, Arg261Thr, and Lys264Gly, and optionally, deletion ofresidues corresponding to Lys263, Asp265 and Arg 266.

It will be understood by those skilled in the art that p40 variants andactive portions thereof that incorporate a D3 variant as describedherein are within the scope of the invention. Similarly, IL-12 proteinsand active portions thereof that contain a p40 variant (IL-12 variants)also are within the scope of the invention. The invention furtherencompasses fusion proteins including IL-12 variants of the inventionand a moiety selected from the group consisting of an antibody moiety, anon-IL-12 cytokine, or an active portion thereof.

Similarly, IL-23 proteins and active portions thereof that contain a p40variant (IL-23 variants) also are within the scope of the invention. Theinvention further encompasses fusion proteins including IL-23 variantsof the invention and a moiety selected from the group consisting of anantibody moiety, a non-IL-23 cytokine, or an active portion thereof.

In another aspect, the invention relates to a nucleic acid that encodesany of the D3 variants, p40 variants, IL-12 variants, and IL-23 variantsof the invention. The invention further encompasses a cell, e.g., aprokaryotic cell, including such a nucleic acid.

The invention also features methods of making such D3 variants, p40variants, IL-12 variants, IL-23 variants and fusion proteins containingthese moieties.

In yet another aspect, the invention provides methods of using thevariants of the invention and the nucleic acids encoding same. Forexample, the invention encompasses a method of treating a patient thatincludes administering to the patient a therapeutically effective amountof a p40 variant of the invention or an active portion thereof.

The foregoing, and other features and advantages of the invention aswell as the invention itself, will be more fully understood from thefollowing figures, description, and claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts the mature amino acid sequence of the α chain, i.e., thep35 subunit, of a mature (wild-type) human IL-12 (SEQ ID NO:1).

FIG. 2 depicts the mature amino acid sequence of the β chain, i.e., thep40 subunit, of a mature (wild-type) human IL-12 (SEQ ID NO:2). DomainD3, corresponding to positions 211-306 (SEQ ID NO:26), is italicized,and the peptide fragment corresponding to positions 258-266 isunderlined (SEQ ID NO:5), with Lys260 and Arg261 highlighted in bold.

FIG. 3 depicts the mature amino acid sequence of the α chain, i.e., thep19 subunit, of a mature (wild-type) human IL-23 (SEQ ID NO:3).

FIG. 4A shows the SDS-PAGE gel for several purified batches of humanIL-12 produced as antibody fusion proteins (lanes 1-8), while FIG. 4Bshows the SDS-PAGE gel for several purified batches of human IL-23produced as antibody fusion proteins (lanes 1-3). The IL-12p35 andIL-23p19 subunit is covalently attached to the antibody heavy chain. The6 kD band is indicated by an arrow. Molecular weights (kD) are indicatedfor the markers (lane M).

FIG. 5 depicts the amino acid sequence of the C-terminal peptidefragment of mature (wild-type) human IL-12p40 subunit. The fragmentstarts at Arg261 (SEQ ID NO:4).

FIG. 6 depicts the amino acid sequence of a peptide fragmentcorresponding to positions 258-266 of a mature (wild-type) humanIL-12p40 subunit (SEQ ID NO:5).

FIGS. 7A and 7B depict an amino acid sequence alignment of IL-12p40subunits from various mammals including human, baboon (Papio anubis),rhesus monkey (Macaca mulatta), mangabey (Cercocebus torquatos), dog(Canis familiaris), cat (Fells catus), horse (Equus caballus), pig (Susscrofa), cow (Bos Taurus), goat (Capra hircus), sheep (Ovis aries), deer(Cervus elaphus), water buffalo (Bubalus bubalis), hamster (Mesocricetusauratus), guinea pig (Cavia porcellus), cotton rat (Sigmodon hispidus),rat (Rattus norvegicus), and mouse (Mus musculus). The two amino acidsin bold indicate the proteolytic cleavage site.

FIG. 8 depicts the amino acid sequence of a variant, referred to hereinas p40V1, of a mature human IL-12p40 subunit (SEQ ID NO:6). Theengineered sequence is underlined.

FIG. 9 depicts the amino acid sequence of a variant, referred to hereinas p40V2, of a mature human IL-12p40 subunit (SEQ ID NO:7). Theengineered sequence is underlined.

FIG. 10 depicts the amino acid sequence of a variant, referred to hereinas p40V3, of a mature human IL-12p40 subunit (SEQ ID NO:8). Theengineered sequence is underlined.

FIG. 11 depicts the amino acid sequence of a variant, referred to hereinas p40V4, of a mature human IL-12p40 subunit (SEQ ID NO:9). Theengineered sequence is underlined.

FIG. 12 depicts the amino acid sequence of a variant, referred herein asp40V5, of a mature human IL-12p40 subunit (SEQ ID NO:10). The alterationis underlined.

FIG. 13 depicts the amino acid sequence of a variant, referred herein asp40V6, of a mature human IL-12p40 subunit (SEQ ID NO:11). Thealterations are underlined.

FIG. 14 depicts the amino acid sequence of a variant, referred herein asp40V7, of a mature human IL-12p40 subunit (SEQ ID NO:12). The alterationis underlined.

FIG. 15 depicts the amino acid sequence of a variant, referred herein asp40V8, of a mature human IL-12p40 subunit (SEQ ID NO:13). The alterationis underlined.

FIG. 16 depicts the amino acid sequence of a variant, referred herein asp40V9, of a mature human IL-12p40 subunit (SEQ ID NO:14). The alterationis underlined.

FIG. 17 depicts the amino acid sequence of a variant, referred herein asp40V10, of a mature human IL-12p40 subunit (SEQ ID NO:15). Thealteration is underlined.

FIG. 18 depicts the amino acid sequence of a variant, referred herein asp40V11, of a mature human IL-12p40 subunit (SEQ ID NO:16). Thealterations are underlined.

FIG. 19 depicts the nucleic acid sequence encoding the full length(wild-type) human IL-12p40 subunit (SEQ ID NO:21).

FIG. 20 depicts the nucleic acid sequence of a synthetic nucleotidefragment, referred to herein as V1V2, encoding portions of p40 variantsp40V1 and p40V2. The V1V2 fragment encompasses a V1 fragment including aregion (underlined) encoding SEQ ID NO:17. This is followed by a linkersequence (lower case), and subsequently, a V2 fragment including aregion (underlined) encoding SEQ ID NO:18.

FIG. 21 depicts the nucleic acid sequence of a synthetic nucleotidefragment, referred to herein as V3V4, encoding portions of p40 variantsp40V3 and p40V4. The V3V4 fragment encompasses a V3 fragment including aregion (underlined) encoding SEQ ID NO:19. This is followed by a linkersequence (lower case), and subsequently, a V4 fragment including aregion (underlined) encoding SEQ ID NO:20.

FIG. 22 depicts the nucleic acid sequence of a synthetic nucleotidefragment, referred herein as V5V6, encoding portions of p40 variantsp40V5 and p40V6. The V5V6 fragment encompasses a V5 fragment including acodon substitution (underlined) encoding Arg261Ala. This is followed bya linker sequence (lower case), and subsequently, a V6 fragmentincluding codon substitutions (underlined) encoding Lys260Ala andArg261Ala.

FIG. 23 depicts the nucleic acid sequence of a synthetic nucleotidefragment, referred herein as V7V8, encoding portions of p40 variantsp40V7 and p40V8. The V7V8 fragment encompasses a V7 fragment including acodon substitution (underlined) encoding Lys260Ala. This is followed bya linker sequence (lower case), and subsequently, a V8 fragmentincluding a codon substitution (underlined) encoding Lys260Gly.

FIG. 24 is a Western blot with a polyclonal anti-hu-p40 antibody.Supernatants of cells transfected with wild-type IL-12p40 and IL-12p40variants (V1-V4) were harvested and processed on an SDS-PAGE gel. Twoindependent clones (a, b) of each IL-12p40 variant p40V1, p40V2, p40V3,and p40V4 were tested. The arrow points to the band of cleaved IL12p40lacking C-terminal fragment (lane p40 wt).

FIG. 25 shows the SDS-PAGE gel for antibody-IL12 fusion proteinscontaining p40 variants p40V1, p40V2, p40V3, p40V4, p40V5, p40V6, p40V7,p40V8, and wild type p40 (lanes 1-9). The upper most main bandrepresents the fusion protein between the antibody heavy chain and thep35 subunit, and the lower main band represents the antibody lightchain. The 6 kDa band was not detected in any of the lanes of thevariant proteins, except lane 5. Molecular weights (kD) are indicatedfor the markers (lane M).

FIG. 26 shows pharmacokinetic data of antibody-IL12 fusion proteinscontaining p40 variants p40V1-p40V8 compared toantibody-(wild-type)-IL12 fusion proteins, administered intravenously.

FIG. 27A shows pharmacokinetic data of antibody-IL12 fusion proteinscontaining p40 variants p40V1-p40V4 compared toantibody-(wild-type)-IL12 fusion proteins (panel A) and FIG. 27B showspharmacokinetic data of antibody-IL12 fusion proteins containing p40variants p40V5-p40V8 compared to antibody-(wild-type)-IL12 fusionproteins (panel B), administered subcutaneously.

FIG. 28 is an IL-12p40 variant having mutations outside the D3 region ofthe protein.

DETAILED DESCRIPTION OF THE INVENTION

The invention describes variants of the cytokine interleukin-12 (IL-12)p40 subunit which have improved stability compared with the wild-typeprotein. In these variants, a region of the p40 subunit which isnormally unstructured and sensitive to proteolytic cleavage is mutatedto be more resistant to proteolytic cleavage. This region, in domain D3,is near the C-terminus of the p40 subunit, encompassing a polypeptidestretch corresponding to amino acids 258-266 in mature human p40(p40(258-266)).

The IL-12p40 subunit is also a component subunit of the cytokineinterleukin-23 (IL-23). IL-23 has two subunits, the α subunit “p19” andthe β subunit “p40.” The p40 subunit of IL-23 is the same as IL-12p40.Therefore, variants of IL-12p40 subunit are likewise variants of theIL-23p40 subunit.

In one general class of embodiments, one or more mutations areintroduced into the region of p40 corresponding to amino acid residues258-266 to eliminate the cleavage site, which in human p40 correspondsto the site between Lys260 and Arg261. In further embodiments, specificmutations are introduced in this region that by modeling generate a moretightly folded structure. In another aspect of the invention, theintroduced mutations additionally are predicted to avoid making theengineered region of the p40 subunit immunogenic. For example, aminoacid substitutions in the engineered region of the p40 subunit arechosen to avoid the creation of peptides that may be recognized aspotential T-cell epitopes in humans.

Mutations may be introduced into the region of p40 corresponding toamino acid residues 258-266 by a variety of mechanisms. For example, inone embodiment, a mutation or mutations are introduced by substitutionof one amino acid residue for another. In a further embodiment, amutation or mutations are introduced by deletion of one or more residuesof the p40 subunit. In yet another embodiment, a mutation or mutationsare introduced by insertion of one or more amino acid residues into thep40 subunit.

In one embodiment, the p40 variants of the invention are contained inprotein compositions such as IL-12 proteins, IL-12 fusion proteins,IL-23 proteins, IL-23 fusion proteins or p40 homodimers. For example, inone embodiment, an IL-12 protein contains a p35 subunit and a p40variant according to the invention. In another embodiment, the IL-23protein contains a p19 subunit and p40 variant according to theinvention. In a further embodiment, a fusion protein contains anantibody portion fused to IL-12 containing an IL-12p40 variant. In aneven further embodiment, a fusion protein contains an antibody portionfused to IL-23 containing an IL-12p40 variant. In a further embodiment,the antibody portion of the fusion protein is an intact antibody, an Fcregion, an sc-Fv, an antigen binding portion of an antibody, or anactive fragment of an antibody. In yet another embodiment, a fusionprotein according to the invention includes an IL-12p40 variant fused toan non-IL-12 or non-IL-23 cytokine or active portion thereof.

In a further aspect of the invention, protein compositions that containone of the p40 variants of the invention have a longer circulatinghalf-life than the corresponding wild-type protein. Thus, in comparisonwith IL-12 or IL-23 proteins that contain the wild-type p40 subunit,IL-12 variants or IL-23 variants that include an engineered p40 subunitof the invention have improved properties as therapeutic agents withregard to their production, formulation and pharmacokinetics.

In a further embodiment, mutations to the IL-12p40 amino acid sequenceare introduced outside the IL-12p40(258-266) region and optionallyoutside the IL-12p40 D3 domain of IL-12p40. Such mutations can beintroduced elsewhere in the D3 domain of IL-12p40 and can be introducedin the other domains. For example, FIG. 28, depicts a sequence of a p40subunit of IL-12 that has alterations in the amino acid sequence outsideof residues 258-266. Leong et al., (2003), Proc. Natl. Acad. Sci. USA,100:1163-1168, the contents of which are incorporated by referenceherein, teach various residues which may be mutated in the IL-12 p40amino acid sequence beyond the 258-266 region. Further, because thethree dimensional crystal structure of IL-12p40 is known (Yoon et al.,(2000), EMBO J., 19:3530-3541, the contents of which are incorporated byreference herein), and residues essential to the interaction of the p40subunit with the p35 subunit of IL-12 are know, selection of othermutations that will not destroy functionality of the p40 subunit aredeterminable by one of skill in the art.

Determination of the Cleavage Product

The invention rests in part on the observation that the IL-12p40 subunitis susceptible to a specific proteolytic cleavage event, and on novelexperimental results defining the cleavage site. It was found that apurified recombinant IL-12 protein, produced in NS/0 cells as describedin Example 2, consistently exhibited a degree of heterogeneity when therecombinant protein was separated by electrophoresis on an SDS-PAGE gelunder reducing conditions, clearly visible as an additional protein bandof approximately 6 kD molecular weight. A similar observation was madefor recombinant IL-23 protein. This is illustrated in FIG. 4 which showsthe SDS-PAGE gel for several purified batches of human IL-12 (Panel A)and human IL-23 protein compositions (Panel B) produced as antibodyfusion proteins.

The contaminant was purified and its amino acid sequence was determined,as described in Example 3, and was found to correspond to the sequenceof the C-terminal 46 amino acid fragment of the IL-12p40 subunit itself,generated by proteolytic cleavage between Lys260 and Arg261 (FIG. 5-SEQID NO:4). Cleavage between Lys260 and Arg261 appears to be highlyfavored despite a prevalence of basic amino acids in the regionsurrounding the cleavage site ( . . . KSKREKKDR . . . (FIG. 6-SEQ IDNO:5) where Lys260 and Arg261 are indicated in bold). However, in spiteof being cleaved from the p40 subunit, the C-terminal peptide fragmentremains non-covalently associated with the rest of the p40 subunit,making it difficult to remove the resulting heterogeneity by furtherpurification of the recombinant protein.

IL-12p40 Protein

The mature human p40 subunit is a 306 amino acid protein resembling asoluble class I cytokine α receptor, composed of domains D1, D2 and D3.The cleavage site (between Lys260 and Arg261) is within D3, afibronectin type III domain of 96 amino acids encompassing the regionfrom 1211 to S306. The sequence for mature human IL-12 p40 subunit isshown in FIG. 2. The amino acid sequence for the D3 is shown in FIG. 2in italics. In the published X-ray crystallographic structure of thehuman IL-12 heterodimer (Yoon et al. (2000), EMBO 1, 19: 3530-41) aportion of a loop within the region of human p40(258-266) of the D3domain is not resolved. Without wishing to be bound by theory,unresolved regions in crystal structures are often an indication offlexible, unstructured loops, and may constitute target sites forproteolysis.

A primary structure alignment of the mature p40 subunit from a varietyof mammalian species is shown in FIGS. 7A and 7B, including human; theprimates baboon, rhesus monkey and mangabey; dog; cat; horse; pig; theruminants cow, goat, sheep, deer, water buffalo; and the rodentshamster, guinea pig, cotton rat, rat and mouse. In the alignment theregion around P40(258-266) exhibits sequence variability, particularlywith respect to its length. Particularly, in some species, notablyruminants, the sequence is shorter, and in others, such as in rodents,the sequence in some instances may contain an additional insert. In manyspecies, the dipeptide motif corresponding to human p40 K260R261 isconserved, either identically or exhibiting two basic amino acids. Thesespecies include agriculturally and commercially important speciesincluding, but not limited to, the horse, cow, goat, pig, and sheep.Consequently, introducing one or more mutations into the region ofnon-human IL-12p40 corresponding to residues 258-266 of human IL-12p40that are analogous to mutations taught herein with respect to humanIL-12p40 may prove useful in reducing or eliminating proteolyticcleavage of non-human IL-12p40 at the K260R261 cleavage site.

In principle, according to the invention, it is possible to use aIL-12p40 variant from a species that lacks a positively chargeddipeptide motif corresponding to human IL-12p40 K260R261 in a human orother heterologous organism. However, in practicing the invention, it isimportant to note that non-human forms of IL-12p40 will generally leadto anti-p40 antibodies when administered to humans. More broadly, it isnot optimal to administer p40 from one species to another. In addition,the potential of various non-human p40 subunits to be proteolyticallycleaved is generally unknown. In addition, p40 subunits from one speciesmay not function in another species, either at the step of assembly withsubunits such as p35 or p19, or at the step of interaction with receptorsubunits.

Variant IL-12p40 Proteins

The invention provides for variant IL-12p40 proteins with mutations inthe D3 domain that improve stability. As used herein, the term “D3variant” refers to a D3 domain of a human p40 subunit of, for example,IL-12 or IL-23, having one or more amino acid alterations as compared towild-type D3. The term “p40 variant” is used herein to refer to a humanp40 subunit of, for example, IL-12 or IL-23, with mutations in the D3domain, i.e., a p40 subunit containing a D3 variant. The term “IL-12variant” is used herein to refer to a human IL-12 protein containing ap40 variant. The term “IL-23 variant” is used herein to refer to a humanIL-23 protein containing a p40 variant.

According to one embodiment of the invention, the D3 domain of a p40variant has at least 70% or more sequence identity with the D3 domain ofwild-type IL-12p40. In a further embodiment, the D3 domain of a p40variant has at least 75% or more sequence identity with the D3 domain ofwild-type IL-12p40. In yet another embodiment, the D3 domain of a p40variant has at least 80% or more sequence identity with the D3 domain ofwild-type IL-12p40, while in further embodiments, the D3 domain of a p40variant has at least 81% or more, or at least 82% or more, or at least83% or more, or at least 84% or more, or at least 85% or more, or atleast 86% or more, or at least 87% or more, or at least 88% or more, orat least 89% or more, or at least 90% or more, or at least 91% or more,or at least 92% or more, or at least 93% or more, or at least 94% ormore, or at least 95% or more, or at least 96% or more, or at least 97%or more, or at least 98% or more, or at least 99% or more identity withthe D3 domain of wild-type IL-12p40.

According to another embodiment of the invention, the amino acidsequence of a p40 variant has at least 70% or more sequence identitywith the amino acid sequence of mature wild-type IL-12 p40. In a furtherembodiment, the amino acid sequence of a p40 variant has at least 75% ormore sequence identity with the amino acid sequence of mature wild-typeIL-12p40. In yet another embodiment, the amino acid sequence of a p40variant has at least 80% or more sequence identity with the amino acidsequence of mature wild-type IL-12p40, while in further embodiments, theamino acid sequence of a p40 variant has at least 81% or more, or atleast 82% or more, or at least 83% or more, or at least 84% or more, orat least 85% or more, or at least 86% or more, or at least 87% or more,or at least 88% or more, or at least 89% or more, or at least 90% ormore, or at least 91% or more, or at least 92% or more, or at least 93%or more, or at least 94% or more, or at least 95% or more, or at least96% or more, or at least 97% or more, or at least 98% or more, or atleast 99% or more identity with the amino acid sequence of maturewild-type IL-12p40.

To determine the percent identity between two amino acid sequences, thesequences are aligned for optimal comparison purposes (e.g., gaps can beintroduced in the sequence of a first amino acid sequence for optimalalignment with a second amino acid sequence). The percent identitybetween the two sequences is a function of the number of identicalpositions shared by the sequences (i.e., % identity=(# of identicalpositions/total # of positions)times 100). If the sequences beingcompared are of unequal length, the shorter of the sequences is used todetermine the total number of positions. The determination of percentidentity between two sequences can also be accomplished using amathematical algorithm.

A non-limiting example of a mathematical algorithm utilized for thecomparison of two sequences is the algorithm of Karlin and Altschul,(1990) Proc. Natl. Acad. Sci. USA, 87:2264-68, modified as in Karlin andAltschul, (1993) Proc. Natl. Acad. Sci. USA, 90:5873-77. Such analgorithm is incorporated into the NBLAST and XBLAST programs ofAltschul et al., (1990) J. Mol. Biol., 215:403-10. BLAST nucleotidesearches can be performed with the NBLAST program, score=100,wordlength=12. BLAST protein searches can be performed with the)(BLASTprogram, score=50, wordlength=3. To obtain gapped alignments forcomparison purposes, Gapped BLAST can be utilized as described inAltschul et al., (1997) Nucleic Acids Research, 25(17):3389-3402. Whenutilizing BLAST and Gapped BLAST programs, the default parameters of therespective programs (e.g., XBLAST and NBLAST) can be used.

In one embodiment, the invention provides D3 variants containing analteration that removes the proteolytic cleavage site between Lys260 andArg261. In one embodiment, the amino acid at position Lys260 is mutated.In a more specific embodiment, Lys260 is replaced with a non-basic aminoacid. Non-basic amino acids include, for example, Ala, Asn, Asp, Cys,Glu, Gln, Gly, Ile, Leu, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val. Forexample, Lys 260 is replaced with either Ala, Asn, Asp, Cys, Glu, Gln,Gly, Ile, Leu, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val. In analternate embodiment, Lys260 is replaced with selenocysteine. Examplesof D3 variants where Lys260 has been replaced by another amino acid areshown in FIGS. 12, 13, 14, 15, 16, and 18.

In another embodiment, Arg261 is mutated. For example, in oneembodiment, Arg 261 is replaced with any non-basic amino acid. Forexample, in one embodiment, Arg261 is replaced with Ala, Asn, Asp, Cys,Glu, Gln, Gly, Ile, Leu, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val. Inan alternate embodiment, Arg261 is replaced with selenocysteine.Examples of D3 variants where Arg261 has been replaced by another aminoacid are shown in FIGS. 12, 13, 17, and 18. In a further embodiment,Lys260 and Arg261 are mutated. For example, in one embodiment, Lys260and Arg261 are each replaced with another amino acid. For exampled,Lys260 is replaced with either Gln, Ala, Asn, or Gly and Arg261 isreplaced with either Ala, Asp, or Thr. Examples of D3 variants whereboth Lys260 and Arg261 have been replaced by other amino acids are shownin FIGS. 13 and 18.

In addition, Lys260 and Arg261 are deleted in one embodiment, while in afurther embodiment, an amino acid is inserted between Lys260 and Arg261

In a further embodiment, a D3 variant is created in which one or more ofLys258, Ser259, Lys260, Arg261, Lys263, Lys264, or Arg266 is eachreplaced by another amino acid. In one embodiment, one or more ofLys258, Ser259, Lys260, Arg261, Lys263, Lys264, or Arg266 is eachreplaced by a non-basic amino acid. For example, in one embodiment,Lys258 is replaced with Gln. In another embodiment, Ser259 is replacedwith Asp. In another embodiment, Lys260 is replaced with Ala, Gly, Asnor Gln. In a further embodiment, Arg261 is replaced with Ala, Thr orAsp. In yet another embodiment, Lys263 is replaced by Ser. In a furtherembodiment, Lys264 is replaced by Gly. In one embodiment, Arg266 isreplaced with Gln, Asp, Asn, or Thr. In a further embodiment, Lys263 andLys264 are replaced each replaced by another amino acid. For example, inone embodiment Lys263 and Lys264 are each replaced by Ser and Glyrespectively. In yet another embodiment, Lys258, Ser259, Lys260, Arg261,Lys263, and Lys264 are replaced by Gln, Asp, Gln, Asp, Ser and Glyrespectively.

In a further embodiment, a D3 variant is created in which one or more ofSer259, Lys260, and Arg261 are each replaced by another amino acid. In afurther embodiment, one or more of Ser259, Lys260, and Arg261 are eachreplaced by a non-basic amino acid. For example, in one embodiment,Ser259, Lys260, and Arg261 are replaced by Asp, Asn, and Thrrespectively.

In another embodiment, Lys258, Ser259, Lys260, and Arg261 are eachreplaced by another amino acid. For example, in one embodiment, Lys258,Ser259, Lys260, and Arg261 are each replaced by a non-basic amino acid.In one embodiment, Lys258, Ser259, Lys260, and Arg261 are each replacedby Gln, Asp, Gln, and Asp respectively. In a further embodiment, Ser259,Lys260, Arg261 and Lys264 are replaced by Asp, Asn, Thr, and Glyrespectively. In yet a further embodiment, Ser259, Lys260, Arg261 andLys264 are replaced by Asp, Asn, Thr, and Gly respectively, whileLys263, and Asp265 are deleted. In yet another embodiment, Ser259,Lys260, and Arg261 are each replaced by Asp, Asn, and Thr respectively,while Lys263, Lys264, and Asp265 are deleted.

Without wishing to be bound by theory, deletions are believed to havethe effect of reducing the conformational flexibility of thep40(258-266) region, thus reducing the ability of the Lys260-Arg261motif to adopt a conformation that allows cleavage by the relevantprotease. Therefore, in one embodiment, one or more of Lys258, Ser259,Lys260, Arg261, Lys263, Lys264, Asp265, or Arg266 is deleted.

In a further embodiment, a D3 variant is created in which one or more ofLys263, Lys264, Asp265, or Arg266 is deleted. For example, in oneembodiment, Lys263 and Asp265 are deleted, while in another embodiment,Lys263, Lys264, and Asp265 are deleted. In another embodiment, Lys263,Lys264, and Asp265 are deleted and replaced by one or more non-basicamino acids. In a further embodiment, Lys263, Lys264, Asp265, and Arg266are deleted. In a further embodiment, one or more of Lys263, Lys264,Asp265 or Arg266 is deleted, while one or more of Ser259, Lys260, orArg261 is replaced by another amino acid. For example, in oneembodiment, Ser259, Lys260, and Arg261 are replaced by Asp, Asn, and Thrrespectively while Lys263, Lys264 and Asp265 are deleted. In a furtherembodiment, Ser259, Lys260, and Arg261 are replaced by Asp, Asn, and Thrrespectively while Lys263, Lys264, Asp265, and Arg266 are deleted.

In other embodiments, the amino acid substitutions are selected suchthat they avoid creating novel T-cell epitopes. Methods to analyzepeptide sequences for their potential to create T-cell epitopes are wellknown in the art (see, e.g., U.S. Patent Application Publication No.2003/0153043; International Publication No. WO 00/034317; and Sturnioloet al. (1999), Nature Biotech., 17: 555-61). In one embodiment, thesequence of human IL-12p40(258-266) is replaced by the sequence KDNTER(SEQ ID NO:17). In other words, Ser259, Lys260, and Arg261 were replacedby Asp, Asn, and Thr respectively while Lys263, Lys264, and Asp265 weredeleted such that the resulting sequence from residue 258-263 in thevariant is KDNTER. The resulting IL-12p40 variant is shown in FIG. 8.

In another embodiment, the sequence of human IL-12p40(258-266) isreplaced by the sequence KDNTEGR (SEQ ID NO:18). In other words, Ser259,Lys260, and Arg261 were replaced by Asp, Asn, and Thr respectively whileLys263, Lys264 and Asp265 were deleted and replaced by only a Glyresidue such that the resulting sequence from residue 258-264 in thevariant is KDNTEGR. The resulting IL-12p40 variant is shown in FIG. 9.

In yet another embodiment, the sequence of human IL-12p40(258-266) isreplaced by the sequence QDQDEKKDR (SEQ ID NO:19). In other words,Lys258, Ser259, Lys260, and Arg261 were replaced by Gln, Asp, Gln, andAsp respectively, such that the resulting sequence from residue 258-266in the variant is QDQDEKKDR. The resulting IL-12p40 variant is shown inFIG. 10.

In a further embodiment, the sequence of human IL-12p40(258-266) isreplaced by the sequence QDQDESGDR (SEQ ID NO:20). In other words,Lys258, Ser259, Lys260, Arg261, Lys263, and Lys264 were replaced by Gln,Asp, Gln, Asp, Ser, and Gly respectively such that the resultingsequence from residue 258-266 is QDQDESGDR. The resulting IL-12p40variant is shown in FIG. 11.

In a further embodiment, a D3 variant is contained within an IL-12p40subunit or active portion thereof. By active portions, it is meant thatan IL-12p40 subunit containing a D3 variant has at least 10% activity inone embodiment, at least 20% in another embodiment, at least 30% inanother embodiment, at least 50% activity in another embodiment, atleast 70% activity in another embodiment, at least 75% activity inanother embodiment, at least 80% activity in another embodiment, atleast 90% activity in another embodiment, at least 95% activity inanother embodiment, at least 99% activity in a further embodiment, atleast 100% activity in another embodiment, at least 150% activity in afurther embodiment, at least 200% activity in another embodiment, atleast 300% activity in a further embodiment, at least 400% activity inanother embodiment, at least 500% activity in another embodiment, or atleast 1000% activity in another embodiment, in comparison to thebiological activity of a wild type IL-12p40 moiety.

Proteins Containing IL-12p40 Variants

The IL-12p40 variants may be introduced into protein compositions inplace of wild-type IL-12p40. Examples of biologically active proteincompositions that include IL-12p40 are p40 homodimers, IL-12 and IL-12fusion proteins, and IL-23 and IL-23 fusion proteins. In one aspect ofthe invention, the IL-12 p35/variant p40 heterodimer consists ofseparate polypeptide chains. Alternatively, the IL-12 p35/variant p40heterodimer consists of a single polypeptide chain. In another aspect ofthe invention, the IL-23 p19/variant p40 heterodimer consists ofseparate polypeptide chains. Alternatively, the IL-23 p19/variant p40heterodimer consists of a single polypeptide chain.

In another aspect of the invention, as part of an IL-12 fusion protein,the IL-12 fusion partner can be an antibody moiety or part of anantibody moiety. Useful antibody moieties include ones that target theIL-12 fusion protein to the tumor environment, for example to the tumorcells themselves, or to the necrotic core of a tumor or to thesupporting stroma. In another embodiment of the invention, the fusionpartner is another cytokine. Useful cytokines include, but are notlimited to, IL-2, IL-7, and IL-15.

In another aspect of the invention, as part of an IL-23 fusion protein,the IL-23 fusion partner can be an antibody moiety or part of anantibody moiety. Useful antibody moieties include ones that target theIL-23 fusion protein to the tumor environment, for example to the tumorcells themselves, or to the necrotic core of a tumor or to thesupporting stroma. In another embodiment of the invention, the fusionpartner is another cytokine. Useful cytokines include, but are notlimited to, IL-2, IL-7, and IL-15.

Nucleic Acids Encoding p40 Variants

In a further aspect of the invention, nucleic acids encodingpolypeptides containing p40 variants of the invention are contemplated.Nucleic acids encoding p40 variants of the invention can be constructed,for example, using DNA techniques familiar to those skilled in the art.Exemplary procedures can be found in Example 1.

FIG. 19 depicts the nucleic acid sequence encoding mature human IL-12p40subunit. FIGS. 20-22 depict synthetic nucleotide fragments for encodingexemplary mutations found in p40 variants of the invention.

Methods of Treatment Using p40 Variants

The p40 variants, including fusion proteins and IL-12 proteins or IL-23proteins containing a p40 variant, of the invention are useful asimmunotherapeutic agents, such as for the treatment of a wide variety ofcancers, based on the demonstrated anti-tumor activity of IL-12proteins. For example, p40 variants of the invention can be used,preferably as a heterodimer with p35, in the treatment of cancersincluding but not limited to renal cancer, colon cancer, ovarian cancer,melanoma and T-cell lymphoma, and as an adjuvant for cancer vaccines.p40 variants can also be used as part of a p40/p40 homodimer to reduce aTH 1 response (e.g., a TH 1 response associated with an autoimmunedisease).

Administration

Both IL-12 variants, IL-23 variants, and p40 variants of the inventioncan be incorporated into a pharmaceutical composition suitable foradministration. Such compositions typically comprise IL-12 variant or afusion protein containing an IL-12 variant and apharmaceutically-acceptable carrier. As used herein, the term“pharmaceutically-acceptable carrier” is intended to include any and allsolvents, dispersion media, coatings, antibacterial and antifungalagents, isotonic and absorption delaying agents, and the like,compatible with pharmaceutical administration. The use of such media andagents for pharmaceutically active substances is well known in the art.

A pharmaceutical composition of the invention is formulated to becompatible with its intended route of administration. Examples of routesof administration include parenteral, e.g., intravenous, intradermal,subcutaneous, oral (e.g., inhalation), transdermal (topical),transmucosal, and rectal administration. Solutions or suspensions usedfor parenteral, intradermal, or subcutaneous application can include thefollowing components: a sterile diluent such as water for injection,saline solution, fixed oils, polyethylene glycols, glycerine, propyleneglycol or other synthetic solvents; antibacterial agents such as benzylalcohol or methyl parabens; antioxidants such as ascorbic acid or sodiumbisulfite; chelating agents such as ethylenediaminetetraacetic acid;buffers such as acetates, citrates or phosphates and agents for theadjustment of tonicity such as sodium chloride or dextrose. pH can beadjusted with acids or bases, such as hydrochloric acid or sodiumhydroxide. The parenteral preparation can be enclosed in ampoules,disposable syringes or multiple dose vials made of glass or plastic.

Medicaments that contain IL-12 variants, IL-23 variants, or p40 variantsof the invention can have a concentration of 0.01 or less to 100% (w/w),though the amount varies according to the dosage form of themedicaments.

Administration dose depends on the body weight of the patients, theseriousness of the disease, the particular type of IL-12p40 variantbeing used, and the doctor's opinion. For example, for an IL-12 variantof the invention, it is generally advisable to administer between about0.01 to about 10 mg/kg body weight a day, about 0.02 to about 2mg/kg/day in case of injection, or about 0.5 mg/kg/day. The dose can beadministered once or several times daily according to the seriousness ofthe disease and the doctor's opinion. For an antibody-IL-12 fusionprotein or antibody-IL23 fusion protein containing a IL-12p40 variant ofthe invention, it is generally advisable to administer between about0.001 to about 1 mg/kg body weight per day, about 0.002 to about 0.5mg/kg/day in case of injection, or about 0.1 mg/kg/day. The dose can beadministered once or twice per 2, 3 or 4 week period, according to thenature and seriousness of the disease and the doctor's opinion.

Aspects of invention are further illustrated by the following examples.

EXAMPLES Example 1 Cloning of Variants of Human IL-12p40 Subunits

Nucleic acids encoding p40 variants of the invention, in particular,p40V1 through p40V8 (SEQ ID NOS:6-13), were constructed using standardDNA techniques familiar to those skilled in the art. In essence, a DNAcassette, encoding a fragment that spans the region encompassing themutated amino acid residues and that is bracketed by convenientrestriction sites, was synthesized de novo (Blue Heron Biotechnology,Bothell, Wash.), and substituted for the corresponding fragment ofwild-type sequence contained in an expression plasmid carrying the p40sequence (see, e.g., pNC-p40 in U.S. Pat. No. 6,838,260). The nucleicacid sequence encoding mature (wild-type) human IL-12p40 subunit isshown in FIG. 19. Expression plasmids encoding the p40 variants werethus obtained.

In particular, the nucleic acids encoding p40V1 and p40V2 were generatedas follows. A cloning vector carrying p40V1 and p40V2 DNA cassettes(pBHV1V2), synthesized as a contiguous fragment as shown in FIG. 20, wasdigested with Bpu10 I and either Eco RI/Sca I or Bbs I, generating anEcoR I/Bpu10 I (for V1) and Bbs I/Bpu10 I (for V2) cassette with EcoRI/Bpu10 I compatible ends, respectively. The Sca I digestion wasincluded to eliminate the similarly sized V2 fragment. These purifiedfragments were cloned into a pNC-p40 expression vector in a tripleligation with the appropriate Bpu10 I/Pvu I and Pvu I/Eco RI fragmentsobtained from NC-p40.

An identical approach was used to generate nucleic acids encoding p40V5and p40V6, starting with the synthesized sequence shown in FIG. 21, andto generate nucleic acids encoding p40V7 and p40V8, starting with thesynthesized sequence shown in FIG. 23.

Similarly, to generate nucleic acids encoding p40V3 and p40V4, theplasmid (pBHV3V4) carrying the synthesized sequence shown in FIG. 22,was digested with EcoR I/Bbs I/Sca I (the Sca I digestion was includedto eliminate the similarly sized V4 fragment), or with Bbs I alone,generating an EcoR I/Bbs I (for V3) and a Bbs I (for V4) cassette,respectively, each with ends compatible with the EcoR I/Bbs I digestedexpression plasmid pNC-p40. Note that for the Bbs I restriction enzymethe recognition and cleavage sequences are separate, and thus sequencescontaining multiple Bbs I recognition sites may generate different,sequence-specific overhangs. The V3 and V4 cassettes were then gelpurified and ligated, respectively, into the expression plasmid pNC-p40in a triple ligation using the appropriate Bbs I/Pvu I and Pvu I/Eco RIfragments obtained from pNC-p40.

The same general approach may be used to generate further nucleic acidmolecules encoding other p40 variants contemplated by the invention.

Example 2 Expression of p40 Variants and of Antibody-IL12; FusionProteins Containing p40 Variants

Standard methods were used to generate cell lines expressing p40variants of the invention (see U.S. Pat. No. 6,838,260). The pNC-p40expression plasmids encoding p40 variants were electroporated intocells, e.g., NS/0 cells. The cells were plated, and transfected cellswere selected on a G418-containing medium. Culture supernatants fromdrug-resistant clones were assayed for production of p40 by ELISA, andthe highest producers were subcloned and tested for stable expression.

To generate antibody-IL-12 fusion protein expressing cell lines with p40variants of the invention, the sequential transfection approachdescribed in U.S. Pat. No. 6,838,360 was followed. For example, thefusion protein DI-NHS-IL12p40V1 was obtained by further transfecting thecell line expressing p40V1 with a second plasmid,pdHL10lambdaDI-NHS-p35, which encodes the NHS76 antibody, wherein theC-terminus of the heavy chain constant region is connected to theN-terminus of the IL-12 p35 subunit. The expression plasmid pdHL10lambdais a derivative of pdHL7, wherein the encoded light chain constantdomain is a lambda chain. The cells were selected on amethotrexate-containing medium, and stable transfectants expressing theantibody fusion proteins were cloned by standard methods.

Example 3 Purification and Characterization of p40 Variants

To characterize the integrity of p40 variants p40V1, p40V2, p40V3, andp40V4 (SEQ ID NO:6-9), spent cell culture media from duplicatetransiently transfected NS-0 cells expressing these variants werecollected, and processed for a Western blot with a polyclonalanti-hu-p40 antibody, shown in FIG. 24. The control wild-type p40subunit was included as a control (lane 1). It was found that thecleaved species lacking the C-terminal 6 kDa fragment, which is normallywell-resolved from the intact p40 species by these electrophoreticconditions (see arrow pointing to band in lane 1), was not present inany of the variants tested (lanes 2-9), and only intact p40 could bedetected. Thus, the tested p40 variants were resistant to a proteolyticactivity present during protein expression.

Antibody fusion proteins containing IL-12p40 variants were purified fromcell culture supernatant using standard techniques based on Protein Acapture (see U.S. Pat. No. 6,838,260).

SDS-PAGE gel of the purified antibody fusion proteins from NS-0 stableclones of the p40 variants given in SEQ ID NOS:6-13 (lanes 1-8) and ofthe non-mutated control (lane 9) is shown in FIG. 25. The middle of thethree major bands represents the non-cleaved p40 subunit, with the faintband slightly above indicating more glycosylated species. The upper mostmain band represents the fusion protein between the antibody heavy chainand the p35 subunit, and the lower main band represents the antibodylight chain. It was found that, with the exception of the sample of theantibody fusion protein containing IL-12p40V5, the 6 kDa band was notpresent. For IL-12p40V5, a residual 6 kDa band was observed (lane 5).

Example 4 Characterization of a Proteolytic Cleavage Site in theWild-Type IL-12p40 Subunit

The identity of the contaminating approximately 6 kDa protein fragmentwas determined by standard methods. Briefly, purified DI-NHS-IL12protein was denatured and reduced in a buffered 6 M guanidine/1 mM DTTsolution at 55° C., and subjected to reverse phase HPLC separation overa Vydac C4 column with a 10% to 90% acetonitrile gradient. The fractioncorresponding to the unidentified peptide species was collected, driedand re-suspended to run on an SDS-PAGE gel confirming that itcorresponded to the 6 kDa fragment, and to determine the sequence of thepeptide by N-terminal sequencing. The sequencing analysis revealed apeptide with the sequence REKKDRVFTD, which corresponds to a sequence inthe mature (wild-type) human IL-12p40 subunit beginning at Arg261.

Example 5 Bioactivity of IL-12 Proteins Containing p40 Variants

Bioactivity of IL-12 proteins containing p40 variants was measured byinduction of IFNγ from human PBMC. The antibody fusion proteins Ab-IL-12containing variants p40V1 to p40V8 were compared to Ab-IL-12 withwild-type p40 and a recombinant human IL-12 protein.

The IFNγ induction assay was performed essentially as described inGately et al. (1995), Current Protocols in Immunology, Section 6.16.4,and Kobayashi et al. (1989), J. Exp Med., 170: 827-845. PBMCs werecultured with PHA-P for 3 days and then 25 IU/ml of hu IL-2 (R&DSystems, Minneapolis Minn.) was added for an additional 24 hours. Thecells were washed, 20 IU/ml of IL-2 was added to all cells, followed byaddition of IL-12 fusion proteins, with a series of two-fold dilutionstarting at 20 ng/ml (in terms of relative mass contribution of IL-12 tothe molecule). Twenty-four hours later, the concentration of IFNγ wasmeasured by ELISA using antibody pairs purchased from R&D Systems.

The results of two separate experiments using PBMCs from differentdonors are summarized Table 1.

TABLE 1 Bioactivity of Ab-IL12 variants in a IFNγ induction assay. IFNγInduction ED50 (ng/ml) Protein AVG (n = 3) SD Exp I R&D IL-12 0.04 0.02Ab-IL12 0.43 0.21 Ab-IL12 V1 1.11 0.59 Ab-IL12 V2 1.09 0.32 Ab-IL12 V31.09 0.21 Ab-IL12 V4 1.44 0.48 Exp II R&D IL-12 0.05 0.03 Ab-IL12 0.530.42 Ab-IL12 V1 1.53 0.58 Ab-IL12 V4 1.79* 0.80 Ab-IL12 V5 0.58 0.39Ab-IL12 V6 1.63 1.46 Ab-IL12 V7 0.99 1.27 Ab-IL12 V8 0.74 0.68 *(n = 2)

Compared to recombinant hu IL-12, the activity of Ab-IL12 with wild typep40 was about 10 fold reduced. It was found that the antibody-IL12variant proteins tested did not significantly further affect theactivity of the protein. Ab-IL12p40V1 to Ab-IL12p40V8 had somewhatreduced activity (approximately 1.5-3 fold less) compared to thecorresponding wild-type antibody-IL-12 fusion protein.

Example 6 Pharmacokinetics of IL-12 Proteins Containing p40 Variants

The pharmacokinetics of the antibody fusion proteins containing the p40variants was determined. The experiments were performed using standardtechniques familiar to those skilled in the art. Briefly, BALB/c mice(n=3 per treatment group) were injected with 25 μg of Ab-IL12 or Ab-IL12variants containing the variants p40V1, p40V2, p40V3, p40V4, p40V5,p40V6, p40V7 and p40V8 in a volume of 0.2 ml, either intravenously inthe tail vein or subcutaneously. At various time points up to 24 hoursand up to 96 hours, respectively, small volumes of blood was taken byretro-orbital bleeding and collected in heparin-coated tubes to preventclotting. After centrifugation to remove cells, the plasma was assayedby capture with anti-human IgG H&L antisera and detection with ananti-human IL-12 antibody. Results were normalized to the initialconcentration in the plasma of each mouse taken within 30 seconds afterinjection (t=0).

FIG. 26 is a compilation of representative experiments assessing thepharmacokinetics of intravenously administered protein. It was foundthat compared to the wild-type control protein, antibody-IL-12 fusionproteins containing variants p40V1 and p40V2, p40V3, p40V4, as well asp40V6, had significantly improved pharmacokinetic values. Particularly,it was found that the half-life of the distribution phase (alpha phase)approximately doubled, and correspondingly the AUC of the variantproteins approximately doubled as well. However, the elimination phase(beta phase) of all Ab-IL-12 fusion proteins remained substantiallysimilar.

These results were consistent with the pharmacokinetics of theseproteins when administered to the mouse subcutaneously FIG. 27, panel A,shows a comparison of wild-type Ab-IL12 with Ab-IL12 variants containingp40 V1, p40 V2, p40 V3, and p40 V4, and panel B shows a comparison ofwild-type Ab-IL12 with Ab-IL12 variants containing p40 V5, p40 V6, p40V7, and p40 V8.

Example 7 Treatment of a Human Patient with IL-12p40 Variants

The IL-12p40 variants of the invention are used to prevent and treathuman diseases and disorders as follows. In general, the preferredmethod of administration is by i.v. infusion or i.v. injection, or bysubcutaneous injection, inhalation, although oral delivery, and othermethods are also possible.

A patient with advanced metastatic prostate cancer, with a history oftreatment by conventional chemotherapy, is treated as follows with anantibody-IL12 fusion protein containing an IL-12p40 variant. The dose ofthe antibody-IL12 fusion protein per treatment cycle is about 150micrograms per kg of body weight, and may be delivered on a single dayor on two or three adjacent days, with administration by drip infusion.Treatment may be combined with a standard-of-care treatment for prostatecancer as determined by a physician as appropriate for the patient.Non-steroidal anti-inflammatory drugs, for example Naproxen™, are alsoprescribed. Treatment cycles are repeated about once every three weeks.

A patient with hormone-refractory breast cancer is treated by dripinfusion with an antibody-IL12 fusion protein containing IL-12p40variant. Non-steroidal anti-inflammatory drugs, for example Naproxen™are also prescribed.

In an alternative treatment strategy, a patient with advancedhormone-refractory prostate cancer or advanced hormone-refractory breastcancer is treated with antibody-IL12 fusion protein containing anIL-12p40 variant about once every three weeks, in combination with anIL-2-containing immunocytokine such as KS-IL2. These two agents may beco-administered by drip infusion. Prior to the treatment, the patient isdosed with an immunostimulatory amount of cyclophosphamide.Non-steroidal anti-inflammatory drugs, for example Naproxen™ are alsoprescribed.

A patient with rheumatoid arthritis is treated with an Fc-p40 fusionprotein, in which the p40 subunit is an IL-12p40 variant, about onceevery two weeks at a dose of about 8 mg/kg, with administration by dripinfusion. Progression of joint destruction is found to be significantlyinhibited by monotherapy, even when compared to disease-modifyinganti-rheumatic drugs.

Equivalents

The invention may be embodied in other specific forms without departingfrom the spirit or essential characteristics thereof. The foregoingembodiments are therefore to be considered in all respects illustrativerather than limiting on the invention described herein. Scope of theinvention is thus indicated by the appended claims rather than by theforegoing description, and all changes which comes within the meaningand range of equivalency of the claims are intended to be embracedtherein.

1. A nucleic acid encoding a variant of an IL-12p40 protein, the variantbeing at least 90% identical to SEQ ID NO:2 and comprising an amino acidalteration at one or more positions corresponding to residues 258-266,wherein the amino acid alteration comprises an amino acid substitutionselected from the group consisting of Lys260Asn, Lys260Gln, andLys260Gly.
 2. The nucleic acid of claim 1, wherein the variant comprisesone or more amino acid substitutions at positions selected from thegroup consisting of Lys258, Ser259, Arg261, Lys263, Lys264, and Arg266.3. The nucleic acid of claim 2, wherein the variant comprises one ormore of Lys258Gln, Ser259Asp, Arg261Ala, Arg261Asp, Arg261Thr,Lys263Gly, Lys263Ser, and Lys264Gly.
 4. The nucleic acid of claim 1,wherein the variant further comprises substitutions at Ser259 andArg261.
 5. The nucleic acid of claim 4, wherein the variant comprisesthe substitutions Ser259Asp, Lys260Asn, and Arg261Thr.
 6. The nucleicacid of claim 4, wherein one or more residues corresponding to Lys263,Lys264, Asp265, and Arg266 are deleted.
 7. The nucleic acid of claim 4,wherein one or more residues corresponding to Lys263, Lys264, Asp265,and Arg266 are substituted by a non-basic amino acid.
 8. The nucleicacid of claim 5, wherein the variant further comprises the substitutionLys264Gly.
 9. The nucleic acid of claim 8, wherein residues Lys263 andAsp265 are deleted.
 10. The nucleic acid of claim 5, wherein residuesLys263, Lys264, and Asp265 are deleted.
 11. A nucleic acid encoding afusion protein comprising the variant of claim 1 and a moiety selectedfrom the group consisting of an antibody moiety, an active fragmentthereof, a non-IL-12 cytokine, and an active portion thereof.
 12. Thenucleic acid of claim 1, wherein the variant is at least 95% identicalto SEQ ID NO:2.
 13. A nucleic acid encoding a variant of an IL-12p40protein, the variant being at least 90% identical to SEQ ID NO:2 andcomprising an amino acid alteration at one or more positionscorresponding to amino acids 258-266, wherein the amino acid alterationcomprises an amino acid substitution at Lys260 and deletion of one ormore of Lys258, Ser259, Lys263, Lys264, and Asp265.
 14. The nucleic acidof claim 13, wherein the variant is at least 95% identical to SEQ IDNO:2.
 15. A nucleic acid encoding a variant of an IL-12p40 protein, thevariant being at least 90% identical to SEQ ID NO:2 and comprising anamino acid alteration at one or more positions corresponding to aminoacids 258-266, wherein the amino acid alteration comprises (i) Lys260Glnor (ii) an amino acid substitution at Lys260 and one or more of aminoacid substitutions Lys258Gln, and Ser259Asp.
 16. The nucleic acid ofclaim 15, wherein the variant further comprises the substitutionLys263Ser and Lys264Gly.
 17. The nucleic acid of claim 15, wherein thevariant is at least 95% identical to SEQ ID NO:2.
 18. A nucleic acidencoding a variant of an IL-12p40 protein, the variant being at least90% identical to SEQ ID NO:2 and comprising an amino acid alteration atone or more positions corresponding to amino acids 258-266, wherein theamino acid alteration comprises amino acid substitution Ser259Asp. 19.The nucleic acid of claim 18, wherein the variant further comprises asubstitution at position Lys260.
 20. The nucleic acid of claim 19,wherein Lys260 is replaced with a non-basic amino acid.
 21. The nucleicacid of claim 20, wherein the non-basic amino acid is Ala, Asn, Gln, orGly.
 22. The nucleic acid of claim 21, wherein the variant is at least95% identical to SEQ ID NO:2.
 23. A nucleic acid encoding a variant ofan IL-12p40 protein, the variant being at least 90% identical to SEQ IDNO:2 and comprising an amino acid alteration at one or more positionscorresponding to residues 258-266, wherein the amino acid alterationcomprises an amino acid substitution selected from the group consistingof Arg261Asp and Arg261Thr.
 24. The nucleic acid of claim 23, whereinthe variant further comprises a substitution at position Lys260.
 25. Thenucleic acid of claim 24, wherein Lys260 is replaced with a non-basicamino acid.
 26. The nucleic acid of claim 25 wherein the non-basic aminoacid is Ala, Asn, Gln, or Gly.
 27. The nucleic acid of claim 26, thevariant being at least 95% identical to SEQ ID NO:2.
 28. An isolatedcell comprising the nucleic acid of claim
 1. 29. An isolated cellcomprising the nucleic acid of claim
 13. 30. An isolated cell comprisingthe nucleic acid of claim
 15. 31. An isolated cell comprising thenucleic acid of claim
 18. 32. An isolated cell comprising the nucleicacid of claim 23.